Determination of lipid, hydrocarbon or biopolymer content in microorganisms

ABSTRACT

A method for determining the content of a bioproduct in a cell culture comprises
         (a) loading a sample of the cell culture onto a density gradient comprising a density determination agent;   (b) centrifuging the product of step (a) for a period of time sufficient to establish a density equilibrium between the cell culture sample and the density gradient;   (c) measuring the density of the cell culture sample containing the bioproduct based on its density equilibrium, and   (d) calculating the weight percent of the bioproduct in the cell culture using the equations:       

       ρ S =( x·ρ   P )+( y·ρ   B ) 
         x+y =1         wherein:   ρ S  represents the density of the cell culture sample containing the bioproduct (in g/mL);   ρ P  represents the density of the bioproduct in pure form (in g/mL);   ρ B  represents the density of the cell biomass in the culture devoid of bioproduct (in g/mL);   x represents the weight % of the bioproduct in the cell culture; and   y represents the weight % of the cell biomass in the cell culture.

BACKGROUND OF THE INVENTION

This invention relates to the determination of the content, in weightpercent, of a chemical substance (hereinafter referred to as a“bioproduct”) in a cell culture using a direct density equilibriummeasurement.

For instance, this method may be used for determining, estimating,and/or tracking the bio-oil or biopolymer content of strains ofmicroalgae and other microorganisms, or in cultures of cellular materialof animal, plant, or insect origin, whose content of such bioproductsmay change with cultivation conditions and/or time, as the case would bein “microorganism lipid induction” industrial processes. The method isalso useful for the direct in situ measurement of storage biopolymeraccumulation in live cells, such as starch in microalgae or plant cellcultures, and of polyhydroxybutyrate or other polyhydroxyalkanoates inphotosynthetic and non-photosynthetic bacteria.

There is widespread interest in the use of microalgae and othermicroorganisms for the generation of renewable biofuels or generation offeedstocks for the pharmaceutical and synthetic chemistry industries. Ofspecific interest to the field is the generation of “bio-oils” or“biodiesel” from the fatty acid components of diacyl- ortriacyl-glycerides. In addition, long-chain terpenoid hydrocarbons,known to naturally accumulate in certain microalgae, e.g. the genusBotryococcus, are of interest to the biofuels and synthetic chemistryindustries. However, there is great variability among differentorganisms in terms of their ability to naturally or artificiallysynthesize and accumulate lipids, hydrocarbons, or polymers. Further,lipid content varies widely during the different stages in the lifecycle of an organism or a culture. Accordingly, there is a need todevelop a method for the quick and reliable in situ assessment of lipid,hydrocarbon or biopolymer content in different microorganisms, and arequirement to be able to spot-check changes inlipid/hydrocarbon/biopolymer content of the cells during the course ofgrowth and/or upon stress of the cultures. This capability is of import,as stress is often applied toward the end of the exponential growthphase to induce lipid accumulation in the living cell.

Microalgae are the organism of choice for the renewable generation ofhydrocarbon-based biofuels. Theoretical fuel production yields frommicroalgae have been estimated to be as high as 4,000 gallons per acrecultivation per year, whereas current yields of soybean oil are onlyabout 50-60 gallons per acre per year. Like other promising biofuels,microalgal oil-production faces many technological barriers that must beovercome before these impressive theoretically maximum yields can beachieved. Developing a simple and direct method for the quantitative insitu determination of bioproduct content, e.g., lipid, hydrocarbon orbiopolymer content, in microalgae and other microorganisms would finduseful application in the screening of a variety of genera and speciesfor such product over-accumulation (microorganism prospecting), as wellas in the monitoring of product content in genetically engineered cellsor in cells of a given culture as a function of growth conditions andexternal treatments.

Density gradient centrifugation using sucrose, Percoll® (a colloidalsilica coated with polyvinylpyrrolidone), or cesium chloride isroutinely employed in biochemical and molecular research to separatedifferent cell types and/or fractionate sub-cellular compartments andmacromolecular complexes on the basis of their differential buoyantdensities independently of particle size or shape. In this approach,continuous or step gradients are cast into transparent centrifuge tubesso that the gradient has a high (bottom) to low (top) concentrationorientation. A range of concentrations of sucrose, Percoll or cesiumchloride is employed, depending on the sedimentation coefficient of thecells or particles investigated. Theoretically, a sucrose gradient mayrange from 80% sucrose at the bottom, to 0% sucrose at the top of thecentrifuge tube, with the density gradient increasing eithercontinuously (continuous gradient) or in discrete increments of 5%, 10%or 20% w/v sucrose (step gradient). Cesium chloride gradients may rangefrom 110% at the bottom, to 0% w/v at the top of the centrifuge tube,permitting attainment of higher density values in the analysis of thebuoyant density of samples. This property of cesium chloride gradientshas been applied in the analysis and separation of DNA samples.

The biological sample is normally layered on top of the gradient andcentrifuged at high acceleration. Depending on their sedimentationcoefficient, or density, samples travel through the gradient until theyreach a point where their density matches that of the surroundingsucrose, Percoll or cesium chloride solution, at which point they willmove no further. The “density equilibrium” properties of a sample dependon its buoyant properties, such that samples found nearest the bottom ofthe gradient will have a relatively high buoyant density, whereassamples found near the top of the gradient will have a relatively lowdensity.

BRIEF SUMMARY OF THE INVENTION

The invention herein comprises a method for determining the content of abioproduct in a cell culture, said method comprising

(a) loading a sample of the cell culture onto a density gradientcomprising a density determination agent;

(b) centrifuging the product of step (a) for a period of time sufficientto establish a density equilibrium between the cell culture sample andthe density gradient;

(c) measuring the density of the cell culture sample containing thebioproduct based on its density equilibrium, and

(d) calculating the weight percent of the bioproduct in the cell cultureusing the equations:

ρ_(S)=(x·ρ _(P))+(y·ρ _(B))

x+y=1

wherein:

ρ_(s) represents the density of the cell culture sample containing thebioproduct (in g/mL);

ρ_(P) represents the density of the bioproduct in pure form (in g/mL);

ρ_(B) represents the density of the cell biomass in the culture devoidof bioproduct (in g/mL);

x represents the weight % of the bioproduct in the cell culture; and

y represents the weight % of the cell biomass in the cell culture.

In one embodiment of the invention the method is conducted a pluralityof times (i.e., two or more times), over a period of time, atappropriate intervals, in order to track the increase or growth ofcontent of the bioproduct in question in the cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the preparation of a sucrose gradient in a Beckman 29×104mm polyallomer centrifuge tube by slow pipetting on the inside wall ofthe inclined tube.

FIG. 2 depicts a sucrose step gradient in the tube of FIG. 1.

FIG. 3 depicts density of sucrose and cesium chloride solutions as afunction of their concentration, measured at 20° C.

FIG. 4 depicts density equilibrium of the cell culture sample from avariety of Botryococcus species.

FIG. 5 depicts buoyant density of live Botryococcus braunii var. Showacell culture sample (a) and that of a sonicated sample (b).

FIG. 6 depicts in vivo buoyant densities of various green microalgae anda cyanobacterium cell culture sample.

FIG. 7 depicts morphology of Chlamydomonas reinhardtii (CC125) cellsprior (a,b) and following sulfur deprivation for 24 h (c,d).

FIG. 8 depicts the effect of sulfur deprivation on the buoyant densityof Chlamydomonas reinhardtii (CC125) cells; control cells (a) and cellsdeprived of sulfur nutrients for a period of 24 h (b).

FIG. 9 depicts buoyant densities in cesium chloride gradient of asulfur-deprived Chlamydomonas reinhardtii (CC125) cell culture sample(a), and of starch grains isolated and purified from these cells (b).

FIG. 10 depicts in vivo buoyant densities of different purplephotosynthetic bacteria.

FIG. 11 depicts in vivo buoyant densities of the purple photosyntheticbacteria Rhodospirillum rubrum as a function of time in sulfurdeprivation.

FIG. 12 depicts buoyant densities of sulfur deprived Rhodospirillumrubrum culture samples (a), and polyhydroxybutyrate (PHB) isolated andpurified from these cultures (b).

FIG. 13 depicts a time-course of buoyant cell density of control andsulfur-deprived Rhodospirillum rubrum cell culture samples as a functionof time under in vivo conditions.

FIG. 14 depicts in vivo buoyant densities of the purple photosyntheticbacteria Rhodospirillum rubrum after sulfur deprivation and densityequilibrium measurement in sucrose (a) and cesium chloride (b) gradientcentrifugation.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises a process as generally described above.

In this work a single-step density gradient centrifugation protocol isused to determine the density of live colonies, single cells andsubcellular compartments under in situ conditions. The gradientcentrifugation method measures the overall density of the sample, fromwhich the bioproduct content of the cell culture sample, correspondingto that of the overall cell culture, is then calculated. The methodprovides quick in situ (intact) cell density measurements for a varietyof samples, including live colonies, intact single cells, cellularfractions and subcellular compartments. In this approach, the absolutebioproduct content of the cells can be calculated. In one embodimentthis method is used for spot-checking bio-oil content in strains ofalgae whose lipid or hydrocarbon content may vary with cultivationconditions and/or time, as the case would be in “lipid induction”experiments. In another embodiment the method is used for spot-checkingbiopolymer content in strains of algae, photosynthetic, andnon-photosynthetic bacteria, as these may accumulate in the course ofgrowth or upon external stress application. In this approach, theun-induced strain may serve as a control for the quantitativecalibration of lipid, hydrocarbon or biopolymer content. In anotherembodiment the method is used to determine the bioproduct content, forexample the lipid, bio-polymer or hydrocarbon content, in amicroorganism at a single point. Examples below pertain to thequantitative measurement of botryococcene hydrocarbons,polyhydroxybutyrate and starch polymer content in a variety ofmicroorganisms. This density equilibrium method also can be applied toprovide insight into the buoyant density of cell walls and thylakoidmembranes in microalgae and photosynthetic bacteria.

The method of the invention may be used for determining the content ofany bioproduct in a cell culture, as long as the density of thatbioproduct is not the same as the cell density. By “bioproduct” is meanta chemical substance that may be formed and/or accumulated by the cellculture. Typical substances whose content may be determined include bothsimple and complex chemicals and/or polymers, including lipids,bio-polymers, hydrocarbons, pharmaceuticals, hormones, biofuels,specialty proteins and other proteins including proteins that areendogenous to the cells but for which the cells over-produce, forexample through genetic engineering or physiological manipulation of thecells. Typically only one bioproduct of interest will be produced andaccumulated by a given cell culture; however, in some situations two ormore bioproducts may be produced and/or accumulated. The cell culturemay comprise living, dormant and/or dead cells, and includes bothmicroorganisms such as algae, bacteria, fungi and yeasts, as well ascell cultures from higher organisms including plant, mammalian, avian,reptilian, fish and insect cell cultures.

In carrying out the process, the densities of the cells per se and thebioproduct whose content is to be determined are ascertained by theuser. This value may already be on hand, for example as provided by asupplier of the cells or of the bioproduct (or in a catalog), or asdetermined on a previous occasion, or it can be measured in the contextof carrying out the process of this invention.

The density of the cell culture containing the bioproduct in question isthen determined using a density gradient centrifugation protocol. Inthis procedure, density gradients of a gradient-determination agent areprepared by dissolving or suspending the gradient-determination agent inwater. The gradient-determination agent may be sucrose, cesium chloride,Percoll, sodium chloride, sorbitol, or any other suitable substance thatmay be employed in such protocols, i.e. a substance that can generatedifferent densities when dissolved or suspended in water. The gradientsmay have any convenient concentration increment. An increment of 10% istypical for such procedures. Each layer contains a single densitygradient increment that is discrete and visibly distinguishable. Then asample of the cell culture containing the substance whose concentrationto be quantified is carefully loaded or layered in the tube on top ofthe gradient. The tubes are then centrifuged for a sufficient period oftime (usually minutes) until a density equilibrium is establishedbetween sample and gradient. All operations can be carried out in thecold room or at room temperature.

EXAMPLES

The following are representative examples of the process of thisinvention. However, they are only illustrative, and are not intended toplace limitations on the invention.

Experimental Procedure

Density gradients of sucrose spanning a concentration range from 10-80%(w/v) and having a concentration increment of 10% were prepared.Similarly, density gradients of cesium chloride spanning a concentrationrange from 35-105% (w/v) and having a concentration increment of 10%were prepared. Sucrose and cesium chloride were dissolved in a solutioncontaining 10 mM EDTA and 5 mM HEPES KOH (pH 7.5). All solutions werekept at 4° C. until use. To pour the gradients, Beckman 29×104 mmpolyallomer centrifuge tubes were stabilized in a rack at a 30-45°angle. Beginning with the highest sucrose or cesium chlorideconcentration, a 4 mL aliquot was carefully pipetted into the centrifugetubes (FIG. 1). Subsequently, 4 mL aliquots of each of the lowerconcentration solutions were carefully pipetted into the centrifugetube, ensuring that the subsequently pipetted solution slowly went downthe side of the tube and layered on top of the preceding aliquot. Thisprocedure was repeated with each of the desired steps in the gradient,entailing the sequential pipetting of 4 mL of 70%, 60%, 50%, 40%, 30%,20% and 10% sucrose or 95%, 85%, 75%, 65%, 55%, 45% and 35% cesiumchloride solutions, respectively. Once the gradient was poured, discretelayers of differing densities could be visually seen, e.g. the sucrosegradient in FIG. 2, which shows the diffraction of light at theinterface of the discrete sucrose gradient steps (10-80% w/v), as wellas the measured distance in cm between the steps in this sucrosegradient. After all sucrose or cesium chloride solutions were set in thegradient, centrifuge tubes were kept at 4° C. until use. The sample,containing colonies, single cells, or subcellular particles of interest,was then carefully layered on top of the preformed gradient, followed bycentrifugation of the polyallomer tubes in a JS-13.1 swing bucketBeckman rotor, at an acceleration of 20,000 g for 30 min at 4° C.

This density equilibrium technique is designed to provide a precisemeasurement of the overall density of the sample. For best visualizationof the resulting bands, gradients were loaded with a 2 mL aliquot of thesample, containing the equivalent of 5 mg dry matter. Dry cell weightanalysis was carried out upon adsorption of the biomass in question, orfiltering the cellular samples through a Millipore Filter (0.22 μm poresize), followed by washing with distilled water. The dry cell weight wasmeasured gravimetrically upon drying the filters at 80° C. for 24 h in alab oven. When applied, disintegration of cellular matter was achievedupon sonication of samples for 4 min with a Branson sonifier, operatedat a Power output of 7 and 50% duty cycle. All such operations werecarried out at 4° C.

Chlamydomonas reinhardtii CC125 cells were sulfur deprived (Melis eta1.2000, “Sustained photobiological hydrogen gas production uponreversible inactivation of oxygen evolution in the green algaChlamydomonas reinhardtii”; Plant Physiol 122: 127-136; Zhang et al.2002 “Biochemical and morphological characterization of sulfur-deprivedand H₂-producing Chlamydomonas reinhardtii (green alga)”; Planta 214:552-561) upon harvesting by centrifugation (5 min, 3,000 g) in themid-exponential stage of growth, followed by washing with sulfur-lackingTAP-S medium (Zhang et al. 2002) and resuspension in TAP-S.Sulfur-deprived (—S) media were made upon substitution of thesulfur-containing salts with their chloride counterparts. Rhodospirillumrubrum cells were anaerobically grown in Ormerod minimal medium, asreported by Melis and Melnicki (2006; “Integrated biological hydrogenproduction”; Int J Hydrogen Energy 31:1563-1573). For the sulfurdeprivation of R. rubrum, cells were harvested by centrifugation, washedand resuspended in Ormerod-S medium. In vivo buoyant densities of thesecells after 49 hours of sulfur deprivation is depicted in FIG. 14.Sucrose gradient centrifugation revealed density equilibrium of ˜70-80%sucrose (ρ=˜1.35 g/mL). CsCl gradient gradient centrifugation revealeddensity equilibrium of ˜45% CsCl (ρ=˜1.35 g/mL).

Densities of sucrose and cesium chloride solutions were calculatedaccording to Bubnik et al. (1995); “Sugar Technologists Manual. Chemicaland physical data for sugar manufacturers and users” (Bartens Pub. Co.,Berlin, Germany) and the CRC Handbook of Chemistry and Physics. 88^(th)ed., Chapter 8, pp. 55-56, 2007, respectively. FIG. 3 shows X-Y plots ofthe sucrose and cesium chloride density parameter ρ (measured in g/mL)as a function of their concentration (% weight per volume) in thesolution, measured at 20° C. Table 1 shows the numerical values in4-decimal points of sucrose concentration (w/v), CsCl concentration(w/v) and their corresponding densities ρ, in g/mL, as used in thiswork.

TABLE 1 Sucrose concentration (w/v), CsCl concentration (w/v) and theircorresponding densitiesρ, in g/mL Sucrose, % w/v ρ, g/mL CsCl, % w/v ρ,g/mL 0.0000 1.0000 0.51000 1.0020 10.000 1.0390 4.1200 1.0293 20.0001.0810 8.5000 1.0625 30.000 1.1280 18.170 1.1355 40.000 1.1780 29.2401.2185 50.000 1.2310 42.040 1.3135 60.000 1.2890 56.910 1.4226 70.0001.3500 79.230 1.5846 80.000 1.4150 107.20 1.7868

Example 1 Cell Density of Botryococcus Species

FIG. 4 compares the density equilibrium properties of different speciesof Botryococcus in sucrose gradient. Botryococcus braunii, var. Yayoiand Botryococcus braunii (UTEX-2441) cells showed a density equivalentto about 60% sucrose or ρ=1.289 g/mL. Botryococcus sudeticus (UTEX-2629)cells proved to have the highest density of the samples examined, havinga density equivalent to 70-75% sucrose (ρ=1.350-1.382 g/mL, FIG. 4 c).On the contrary, cells of Botryococcus braunii, var. Showa, were thelightest among the Botryococci examined, having a density equivalent ofless than the 10% sucrose (p<1.039 g/mL, FIG. 4 d).

Botryococcus braunii, var. Showa are colonial green microalgae, known todiffer from other members of the Chlorococcales in terms of theproduction of high concentrations of liquid hydrocarbons, i.e., C₂₉-C₃₄botryococcenes, that apparently confer to these samples a very lowbuoyant density. In order to test the hypothesis that botryococcenehydrocarbons are indeed the cause of the low overall biomass density ofthese samples, fractionation of the cellular matter was implemented bysonication, followed by sucrose density centrifugation of the crudehomogenate. As seen in FIG. 4 e (B. braunii var. Showa, sonicatedcells), the disintegrated biomass yielded three different densityequilibrium components: a yellow floater band consisting of a mixture ofbotryococcene and carotenoid with an apparent p<1.0 g/mL; a green bandwith a density equivalent to about 10-20% sucrose concentration(ρ=1.039-1.081 g/mL), suggesting the presence of B. braunii cellsdepleted from their botryococcene; and a green band with a densityequivalent to about 45% sucrose concentration (ρ=1.204 g/mL), suggestingthe presence of cells totally free of botryococcene and/or the presenceof thylakoid membranes, apparently originating from the lysis of thecells. This interpretation is consistent with the observation thatresolved thylakoid membranes from Chlamydomonas reinhardtii (CC-503)also had a density equivalent to about 40-45% sucrose concentration(ρ=1.178-1.204 g/mL, FIG. 4 f), and with previous measurements ofthylakoid membrane densities of around 1.17 g/mL.

In order to better define the densities of the Botryococcus braunii var.Showa components, centrifugation of intact and sonicated cells wasconducted with a sucrose gradient covering the 0-10% concentration rangeand having 2% step increments. Intact colonies of Botryococcus brauniivar. Showa were found to have a density equivalent to about 8% sucroseconcentration, which corresponds to ρ=1.031 g/mL (FIG. 5 a). Sonicatedcells released a low-density yellow-colored band that stayed at the topof the sucrose gradient, having a density lower than that of 0% sucrose(ρ<1 g/mL), chemically identified to be a mixture of carotenoid andbotryococcene (not shown). The remainder of the cell debris and thethylakoid membranes all precipitated at the bottom of the centrifugetube, as they had a density greater than that of 10% of sucrose (ρ>1.039g/mL, FIG. 5 b). It is evident that mechanical fractionation andapplication of the density equilibrium principle is necessary andsufficient for the release and separation of botryococcene from the restof the biomass and broken cell matter.

Example 2 Cell Density of Unicellular Green Algae and a Cyanobacterium

A comparative analysis of density equilibrium for various green algaeand a cyanobacterium is given in FIG. 6. Chlamydomonas reinhardtii(CC125) were the heaviest among these samples, having densityequilibrium of about 70% sucrose concentration (ρ=1.350 g/mL, FIG. 6 e).On the other hand, the cell wall-less mutant of Chlamydomonasreinhardtii (CW15, FIG. 6 b) and Dunaliella salina (FIG. 6 a), whichlack the heavy cell wall of the fresh-water microalgae, had lowerdensity equilibrium values. Chlamydomonas reinhardtii (CW15) had densityequilibrium at the interface between 45-50% sucrose (ρ=1.204-1.231 g/mL)while Dunaliella salina equilibrated at the interface between 25-30%(ρ=1.104-1.128 g/mL). It may be concluded that cell walls addsubstantially to the density of cells. Scenedesmus obliquus (freshunicellular green algae, FIG. 6 c) and Synecocystis PCC 6803(cyanobacteria, FIG. 6 d) had about the same density (ρ=1.289 g/mL) asthey equilibrated in the 60% sucrose range.

These results are consistent with reports on the sedimentationproperties of cell wall fractions, which appear to be more dense thancytoplasmic membranes. Flammann et al. (1984; “Characterization of thecell wall and outer membrane of Rhodopseudomonas capsulate” J Bacteriol159(1): 191-198) observed that fragmentation and gradient centrifugationof Rhodopseudomonas capsulatus St. Louis (ATCC 23782) resulted in theseparation of a relatively light cytoplasmic membrane of lipids andproteins (ρ=1.139 g/mL) from a relatively heavier cell wall fraction(ρ=1.215 g/mL) containing primarily peptidoglycans andlipopolysaccharides.

Example 3 Effect of Starch Accumulation on Cell Density

Sulfur deprivation of photosynthetic organisms is known to induce anearly 10-fold increase in starch content, presumably as photosynthesisand metabolism are shifted away from protein and growth, and more towardcarbohydrate biosynthesis. The effect of such substantial biopolymeraccumulation on the density equilibrium properties of Chlamydomonasreinhardtii (CC 125) was investigated in this example.

Induction of starch accumulation by S-deprivation is evident in themorphology of the cells. FIG. 7 (a, b) shows relatively small ellipsoidcontrol cells before (a) and after staining with iodine (b). Iodinestained the polar end of the cell opposite to the flagellae, where thechloroplast is localized, and where starch grains accumulate (FIG. 7 b).Small ellipsoid cells are converted into relatively large sphericalstructures within 24 h of S-deprivation (FIG. 7 c). Staining with iodinerevealed the presence of starch nearly throughout the large sphericalcells (FIG. 7 d), offering evidence of the abundance of starch underthese conditions. Detailed microscopic analysis showed that the densityof starch staining with iodine was maximal after about 24-36 h inS-deprivation and that normally small and ellipsoid C. reinhardtii cellschanged shape and size during this S-deprivation period to become mostlylarger and spherical. This may indicate a cell effort to conserveresources (starch accumulation in the chloroplast) so to be able toquickly recover from the stress conditions (S-deprivation) as soon as itis alleviated.

FIG. 8 shows the result of density equilibrium measurements of controland S-deprived C. reinhardtii. Upon centrifugation in our sucrosegradient, control C. reinhardtii yielded a band at about the interfaceof 70% sucrose concentration (ρ=1.35 g/mL; FIG. 8 a). However, theS-deprived cells quantitatively pelleted at the bottom of the sucrosegradient centrifuge tube (FIG. 8 b), suggesting a density greater thanthat of 80% sucrose concentration (ρ>10.4 g/mL). To obtain a betterestimate of the density equilibrium for the starch-loaded C. reinhardtii(S-deprivation conditions), centrifugation of the latter in a cesiumchloride gradient was undertaken.

FIG. 9 a shows the density equilibrium of S-deprived C. reinhardtii,with a buoyant density of about 55% (w/v) cesium chloride (ρ=1.42 g/mL).FIG. 9 b shows the density equilibrium of purified starch from thesesamples, having a buoyant density of about 85% (w/v) cesium chloride(ρ=1.63 g/mL). Starch polymer accumulation by cells results in a greateroverall density of the biomass, which is opposite to the effect oflipids and hydrocarbons.

Example 4 Effect of Polyhydroxybutyrate (PHB) Accumulation on CellDensity

Sucrose density gradient centrifugation of phototrophically grown cellsof three different photosynthetic bacteria (Rhodospirillum rubrum,Rhodobacterpalustris and Rhodobacter sphaeroides) was applied to measuretheir density equivalents. As seen in FIG. 10, all three species showeddensity equilibrium values of about 55% sucrose concentration (ρ=1.260g/mL) with a minor band at 50% sucrose (ρ=1.231 g/mL). This densityequilibrium of the photosynthetic bacteria is consistently lower thanthat of the fully walled microalgae and cyanobacteria (Table 2). Buoyantproperties of samples investigated are listed on the basis of densityequilibrium, from low to high. Botryococcus braunii (var. Showa) has thelowest density equilibrium value of all microorganisms examined, causedby the constitutive expression and accumulation of liquid hydrocarbons(C30 botryococcene). Dunaliella salina and the CW15 cell wall-lessstrain of Chlamydomonas reinhardtii are relatively lighter than theother green microalgae examined, suggesting that cell walls add to thebuoyant density of the cells. All photosynthetic bacteria examined hadsimilar density equilibrium properties, lower than that of thefreshwater green microalgae. Chlamydomonas reinhardtii (CC125) had thehighest apparent density equilibrium measured in this work. Examinationof the results in Table 2 suggests that such systematic difference canbe directly attributed to the higher density of cell walls in themicroalgae and cyanobacteria over that in the photosynthetic bacteria.The green microalgal and cyanobacterial cell walls are made mostly ofglycoproteins, which are rich in arabinose, mannose, galactose andglucose. Purple photosynthetic bacterial cell walls containpeptidoglycans, carbohydrate polymers cross-linked by protein, and otherpolymers made of carbohydrate protein and lipid. The latter have a lowerbuoyant density than the former.

TABLE 2 Buoyant density of cells by the sucrose or CsCl gradient densityequilibrium method Distance from [Sucrose], Density, Biological samplethe surface, cm % g/mL C39-C34 botryococcene 0.0 0 <1.0 hydrocarbonsBotryococcus braunii 0.7 ~8 1.031 (Showa) Dunaliella salina 2.0 25-301.104-1.128 Isolated thylakoid membranes 3.4 40-45 1.178-1.204Chlamydomonas reinhardtii 3.6 45-50 1.204-1.231 (CW-15) Rhodobacterpalustris 4.1 50-55 1.231-1.260 Rhodobacter sphaeroides 4.1 50-551.231-1.260 Rhodospirillum rubrum 4.1 50-55 1.231-1.260 Scenedesmusobluquus 4.8 ~60 1.289 Synechocystis PCC 6803 4.8 ~60 1.289 Botryococcusbraunii (Yayoi) 5.0 60-65 1.289-1.319 Botryococcus braunii 5.1 60-651.289-1.319 (UTEX-2441) Botryococcus sudeticus 5.7 70-75 1.350-1.382(UTEX-2629) Chlamydomonas reinhardtii 5.5 ~70 1.350 (CC125) Distancefrom Density, Bioproduct the surface, cm [CsCl], % g/mLPolyhydroxybutyrate 2.5 65 1.482 Starch 4.2 85 1.630

The effect of polyhydroxybutyrate polymer accumulation on cell densitywas also investigated. When Rhodospirillum rubrum are subjected toS-deprivation, they accumulate polyhydroxybutyrate (PHB), derived as aproduct of carbon assimilation and serving these photobacteria as anenergy storage polymer, i.e., like starch serves the microalgae in theform of an energy storage compound, to be metabolized as substrate forfast growth when the stress condition is alleviated. Microbialbiosynthesis of PHB starts with the condensation of two molecules ofacetyl-CoA to yield acetoacetyl-CoA, which is subsequently reduced tohydroxybutyryl-CoA. The latter is then polymerized into PHB, which formssizable grains that can be visibly seen under the microscope.

FIG. 11 shows Rhodospirillum rubrum biomass density equilibriummeasurements. FIG. 11 a (0 h in —S) shows cells from control culturesprior to S-deprivation. FIGS. 11 b through 11 e show density equilibriumcharacteristics of cells from cultures that were S-deprived for 6, 13,49 and 59 h, respectively. It is evident that the density equilibrium ofthe cells increases as a function of time in S-deprivation. It issuggested that the cell density increases as PHB accumulates in thecytoplasm of the photobacteria.

For times of incubation longer than 50 h under S-deprivation conditions,R. rubrum pelleted in the sucrose gradient, suggesting a ρ>1.4 g/mL(FIG. 11, 59 h). This is attributed to the increasing amounts of PHB inthese cells. To obtain a more accurate reading of the densityequilibrium of these samples (S-deprivation longer than 50 h) a Cesiumchloride gradient centrifugation was applied. FIG. 12 a shows suchS-deprived R. rubrum having a density equilibrium of 45-55% Cesiumchloride, translating into ρ=1.42 g/mL. FIG. 12 b shows the densityequilibrium of purified PHB on Cesium chloride gradient centrifugation,revealing a 55-65% equilibration (ρ=1.48 g/mL). This p value for R.rubrum PHB is greater than those of Escherichia coli PHB, and ofWautersia eutropha H116 PHB, which were reportedly around 1.25 g/mL(Resch et al. 1998; “Aqueous release and purification ofpoly(beta-hydroxybutyrate) from Escherichia coli.”; J Biotechnol65:173-182; Kobayashi et al. 2005; “Novel intracellular3-hydroxybutyrate-oligomer hydrolase in Wautersia eutropha H16”; JBacteriol 187 (15): 5129-5135). Such discrepancy is probably due to thefact that different organisms produce structurally different PHBgranules with their own density characteristics and physicochemicalproperties.

FIG. 13 shows a quantitative measurement of this phenomenon, revealingphotosynthetic bacterial cell density increase from ρ=1.23 g/mL in thecontrol to ρ=1.43 g/mL in the S-deprived cells, occurring with a halftime of about 20 h. Control cells also increased their density with timein cultivation, albeit more slowly, presumably because they begin toaccumulate PHB as they approach the stationary growth phase.

There is controversy in the literature pertaining to the buoyant densityof cells with and without PHB. We concluded that polymer accumulation inliving cells (starch in C. reinhardtii and PHB in R. rubrum) causes ahigher biomass density. This is clearly seen in these two diversespecies, the unicellular green algae C. reinhardtii, when theyaccumulate starch, and the purple photosynthetic bacteria R. rubrum,when they accumulate PHB. This outcome can be rationalized uponconsideration of the tight packing of carbon, oxygen and hydrogen groupsin these polymers, resulting in a greater overall biomass density. Theeffect of polymers on cell density equilibrium is in sharp contrast tothe accumulation of C30 botryococcene hydrocarbons and, presumably,triglycerides in unicellular green algae, which results in substantiallygreater buoyancy (lower density) of the biomass. The density equilibriumapproach thus can be employed as a quick and reliable method for the insitu determination of biomass density, from which precise estimates ofbioproduct content can be made.

Example 5 In Situ Quantitation of Lipid, Hydrocarbon or BiopolymerContent from the Density Equilibrium Measurement

A system of two equations was devised that permits estimation of the %(w:w) bioproduct (e.g., lipid, hydrocarbon biopolymer, biodiesel, etc.)content in biological samples, based on the density equilibriummeasurement discussed in this work.

ρ_(S)=(x·ρ _(P))+(y·ρ _(B))  (1)

x+y=1  (2)

wherein

ρ_(S) represents the overall density of the cell culture samplecontaining the bioproduct in g/mL;ρ_(P) represents the density of the pure bioproduct in g/mL;ρ_(B) represents the density of the respective biomass, devoid of thebioproduct in g/mL;x represents the % fractional weight of the bioproduct in the cellculture; andy represents the % fractional weight of the biomass, devoid of thebioproduct.

The parameter ρ_(P) would depend on the chemical nature of thebioproduct but it would be independent of the amount accumulating in thesample. Similarly, the parameter ρ_(B) would be constant for a specificcell type or sample but independent of the bioproduct in question. Onthe other hand, the variable ρ_(S) would change as a function of therelative proportion between bioproduct versus biomass and needs to beexperimentally determined during the various stages of growth and/or asa function of stress applied to the organism. Solution of the system ofthese two equations (1 and 2) for x and y permit a fast and quantitativein situ measurement of lipid, hydrocarbon or biopolymer content in smallsamples of living cells.

By way of example, equations (1) and (2) were solved separately forbotryococcene, starch and polyhydroxybutyrate content in theirrespective cell types using results from the density equilibriummeasurements reported above; on the basis of results given in Table 2,PB can be normalized to be equal to 1.30 g/ml for algae and 1.25 g/mlfor purple photosynthetic bacteria. On the other hand ρ_(P) forbotryococcene (0.86 g/ml), PHB (1.48 g/ml) and starch (1.63 g/ml) arealso known. Density equilibrium measurements conducted in this work haveshown ρ_(S) for Botryococcus braunii var. Showa (1.031 g/ml), S-deprivedRhodospirillum rubrum (1.42 g/ml), and S-deprived Chlamydomonasreinhardtii (1.42 g/ml). Solution of eq. (1) and (2) with these measuredvalues yielded estimates of 61.4% botryococcene in Botryococcus brauniivar. Showa, consistent with a 35-85% w/dw. Table 3 presents a summary ofthe calculated w/dw bioproducts content in these various biologicalsamples. The results provide testimony to the validity and utility ofthe density equilibrium method for the quick and precise estimation oflipid/hydrocarbon or biopolymer content in live cells in situ.

TABLE 3 Fraction “x” of bioproduct accumulation (w/dw) in differentmicroorganisms. ρ_(B) ρ_(P) ρ_(S) x Bioproduct Microorganism g/mL g/mLg/mL (%) Botryococcene Botryococcus 1.30 0.86 1.03 61.4 braunii var.Showa Starch Chlamydomonas 1.30 1.63 1.42 36.4 reinhardtiiPolyhydroxybutyrate Rhodospirillum 1.25 1.48 1.42 73.9 rubrum

The foregoing descriptions are offered primarily for purposes ofillustration. Further modifications, variations and substitutions thatstill fall within the spirit and scope of the invention will be readilyapparent to those skilled in the art. All such modifications comingwithin the scope of the appended claims are intended to be includedtherein.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

1. A method for determining the content of a bioproduct in a cellculture, said method comprising (a) loading a sample of the cell cultureonto a density gradient comprising a density determination agent; (b)centrifuging the product of step (a) for a period of time sufficient toestablish a density equilibrium between the cell culture sample and thedensity gradient; (c) measuring the density of the cell culture samplecontaining the bioproduct based on its density equilibrium, and (d)calculating the weight percent of the bioproduct in the cell cultureusing the equations:ρ_(S)=(x·ρ _(P))+(y·ρ _(B))x+y=1 wherein: ρ_(S) represents the density of the cell culture samplecontaining the bioproduct (in g/mL); ρ_(P) represents the density of thebioproduct in pure form (in g/mL); ρ_(B) represents the density of thecell biomass in the culture devoid of bioproduct (in g/mL); x representsthe weight % of the bioproduct in the cell culture; and y represents theweight % of the cell biomass in the cell culture.
 2. A method accordingto claim 1 in which the bioproduct is selected from lipids,hydrocarbons, biofuels, proteins, pharmaceuticals, hormones andbiopolymers.
 3. A method according to claim 1 in which the bioproductcomprises a lipid, hydrocarbon, or bio-oil.
 4. A method according toclaim 1 in which the bioproduct comprises a biopolymer.
 5. A methodaccording to claim 1 in which the cell culture is selected frombacteria, algae, fungi, yeasts, plant cells, mammalian cells, insectcells, reptilian cells, fish cells and avian cells.
 6. A methodaccording to claim 1 in which the cell culture comprises algae.
 7. Amethod according to claim 1 in which the density determination agentcomprises sucrose.
 8. A method according to claim 1 in which the densitydetermination agent comprises cesium chloride.
 9. A method according toclaim 1 further comprising obtaining the cell culture sample from a cellculture and conducting the method a single time.
 10. A method accordingto claim 1 further comprising obtaining a plurality of samples atdifferent times and/or under different conditions from a cell cultureand carrying out the method for each of said samples so as to observethe change in content of said bioproduct over time and/or underdifferent conditions.